The adult heart is a dynamic organ;however cardiomyocytes become terminally differentiated shortly after birth. Over time, adult cardiomyocytes are exposed to multiple pathological stressors that can induce hypertrophic growth and dysfunction. The progression of disease leads to a variety of adaptive responses including the re-expression of fetal proteins. Despite advances in the knowledge of pathways that control hypertrophy, little is known about how modifiers of gene regulation influence anatomical reprogramming of the heart. I hypothesize that histone variants are important regulators that govern changes in gene expression in the diseased and failing heart. To study the changes in chromatin-bound proteins that occur as cardiac disease develops, cardiac hypertrophy was induced in mice by constricting the transverse aorta. Our lab's proteomic analyses identified 1048 proteins from cardiac nuclei including 54 histone variants and revealed distinct alterations in nucleosome stoichiometry which correlate with the manifestation of heart disease at the organ level. Moreover, analysis of individual histone H1 variants-the so-called 'linker histone', responsible for the formation of higher order chromatin structures-revealed distinct alterations of the ratios expressed during cardiac hypertrophy: H1.0 and H1.1 variants increased concomitant with a decrease in the H1.2, H1.3, H1.4 and H1.5 isoforms. We hypothesize that histone H1 variants differentially regulate physically distinct regions of chromatin. We further reason that the ratio of core histones (H2a, H2b, H3 and H4) to linker histone H1 may be an important parameter controlling global chromatin packaging. I propose two aims to test this hypothesis. First, I will determine the cardiac-specific stoichiometry of H1 isoforms using top-down and bottom-up mass spectrometry analyses. I will examine core and linker histone variants to reveal changes in stoichiometry corresponding to the phenotypic presentation of heart disease. Second, I will identify the genomic location of nucleosomes containing cardiac H1 isoforms and determine changes in the occupancy pattern during the development of disease. The H1 variant containing nucleosomes will be selected from nuclei by immunoprecipitation and positioning will be determined by microarrays/DNA sequencing. These studies will provide the first insights into the role of the histone H1 family members in cardiac hypertrophy and will lay the groundwork for a genome-wide interrogation of their in vivo actions. PUBLIC HEALTH RELEVANCE: Heart disease is the leading cause of death in the USA and novel insights into the progression of complications such as heart failure are much needed. This application tests the hypothesis that a family of specific proteins called histone H1 control expression of genes in the normal heart and that alterations in this family underlie heart hypertrophy and failure. These studies will provide the first data on the histone H1 family in the heart including isoform-specificity and details on genomic targets-key insights for future therapeutic approaches.